Composition and methods for treating ischemic wounds and inflammatory conditions

ABSTRACT

Methods, compositions, and treatment protocols for treating ischemic wounds and inflammatory conditions in a patient. The treatment protocols comprise or consist of using a modified collagen gel (MCG) to promote healing of ischemic wounds and reduce inflammation at the wound site and in other inflammatory conditions. The modified collagen gel comprises generally a dispersion of collagens in an aqueous matrix comprising water and glycerine, where the amount of Type I collagen is greater than the amount of Type II and Type III collagens in the gel.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 15/540,661,filed Jun. 29, 2017 as the U.S. National Stage of International PatentApplication No. PCT/US2015/068147, filed Dec. 30, 2015, which claims thepriority benefit of U.S. Provisional Patent Application Ser. No.62/097,734, filed Dec. 30, 2014, entitled Composition and Methods forTreating Ischemic Wounds and Inflammatory Conditions, each of which isincorporated by reference in its entirety herein.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions, methods, and treatmentprotocols for treating ischemic wounds and inflammatory conditions usinga modified collagen gel.

Description of Related Art

Chronic wounds are rarely seen in individuals who are otherwise healthy.In the United States, chronic wounds affect over 6.5 million patients.The burden is also rapidly growing due to increasing healthcare costs,an aging population, and a sharp rise in the incidence of diabetes andobesity worldwide. With the cost of chronic wound care sharply rising,efforts are underway to find simple and inexpensive solutions that maybe applied to a broad group of affected people. Ischemia is caused bylimited blood supply to the wound site causing a shortage of oxygen andother blood-borne products required by the tissue to pay for theincreased metabolic cost of healing. Peripheral vascular disease ordisruption is a common cause of ischemia that may also be viewed asanemia localized to the wound site. Individuals with poor peripheralcirculation are at high risk for developing ischemic wounds. Othermedical conditions also associated with ischemic wounds are diabetesmellitus, renal failure, hypertension, lymphedema, inflammatory diseasessuch as vasculitis or lupus, and current or past tobacco use.

SUMMARY OF THE INVENTION

The present invention is broadly concerned with methods of treating anischemic wound in a patient, where the patient has an ischemic woundsite. The methods generally comprise (or consist of) applying atherapeutically effective amount of a modified collagen gel (MCG)composition to the ischemic wound site to yield a treated ischemic woundsite. The gel is applied for a therapeutically effective period of timesuch that the modified collagen gel promotes healing of the treatedischemic wound site. In general, the modified collagen gel comprises(consists essentially or even consists of) modified collagen of long andshort polypeptides, dispersed in an aqueous matrix comprising water andglycerine. Preferably, the modified collagen is a hydrolyzed bovinecollagen. The modified collagen gel comprises each of Type I, Type II,and Type III collagens, but is primarily Type I and Type III, with TypeI being the most abundant. The modified collagen gel comprisesadditional proteins, including hemoglobin (both alpha and betasubunits), and carbonic anhydrase 2.

The invention is also concerned with use of a modified collagen gelcomposition comprising a dispersion of about 52% by weight hydrolyzedbovine collagen, dispersed in an aqueous matrix comprising water andglycerine in the treatment of an ischemic wound. Preferably, themodified collagen gel comprises a plurality of proteins characterizedaccording to Table 1 below.

Also described herein are methods of treating an inflammatory conditionin a patient suffering from the inflammatory condition. The methodsgenerally comprise (or consist of) administering a therapeuticallyeffective amount of a modified collagen gel composition to the patient,such as to the location of inflammation in the patient. The modifiedcollagen gel is administered for a therapeutically effective period oftime, such that the modified collagen gel reduces the severity of theinflammatory condition in the patient. Administration can includetopical application of the modified collagen gel to a site of dermalinflammation (for example), as well as injection of the modifiedcollagen gel.

The invention is also concerned with use of a modified collagen gelcomposition comprising a dispersion of about 52% by weight hydrolyzedbovine collagen, dispersed in an aqueous matrix comprising water andglycerine in the treatment of an inflammatory condition.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows representative histology images of the treated/untreatedischemic wounds in Example 1.

FIG. 2 shows A: representative mosaic images of wound-edge tissues thatwere immunostained in Example 1; B: a bar graph quantifying macrophageinfiltration at day 7 post wounding in ischemic wounds treated oruntreated with MCG. Data are presented as mean±SEM (n=6); *p<0.05compared with untreated wounds; and C: up-regulation of macrophagemannose receptor 1 (MRC-1) gene expression in THP-1 differentiated humanmacrophages treated with MCG for 24 hour. Data are presented as % changecompared with untreated cells. Data are mean±SEM (n=4); *p<0.05. TD,Tegaderm™; MCG.

FIG. 3 graphs indicating induction of IL-10 and β-FGF genes by MCG;Up-regulation of IL-10 and β-FGF gene expression in THP-1 differentiatedhuman macrophages treated with MCG for 24 hours. The expressions of mRNAfor IL-10 and β-FGF were determined using quantitative real-time PCR.Data are presented as % change compared with untreated cells. Data aremean±SEM (n=4); *p<0.05.

FIG. 4 shows A and B: graphs indicating increased CCR2 (M2c macrophagemarker) expression in macrophages treated with MCG; and C:Representative images of control and treated wound-edge tissuesimmunostained using Anti-MAC387 (macrophages, green) and Anti-CCR2 E68(M2c macrophages, red).

FIG. 5 shows A and B: graphs indicating that MCG promotesvascularization of ischemic wounds; and C: Representativeimmunofluorescence images from wound sections stained with vonWillebrand factor marking vascular endothelial cells (red).

FIG. 6 shows images demonstrating vascularization of MCG-treatedischemic wounds was enhanced by endothelial cell proliferation basedupon representative immunofluorescence images of wound sections (8 μm)at day 21 post wounding stained using Ki67 (marker of proliferatingcells, green) and von Willebrand factor (endothelial cells, red)antibodies.

FIG. 7 shows data demonstrating vascularization of MCG-treated ischemicwounds displayed improved maturity and functionality. A: Wound tissuesections immunostained with an antibody against von Willebrand factor(red) followed by counterstaining with DAPI (blue), viewed with aconfocal laser scanning microscopy at a 1,000× magnification. Z-stackimages were created by merging serial scans of thick tissue section (20μm); and B: Laser Doppler images of ischemic flaps on the back of thepig at day 21 post wounding.

FIG. 8 shows data demonstrating increased vimentin expression inischemic wounds treated with MCG. A: Mosaic images of wound tissuesections that were immune-stained with antivimentin (red) and DAPI(blue). (B) Bar graph shows quantitation of vimentin expression at day21 post wounding in MCG-treated or -untreated ischemic wounds. (C)Real-time PCR was used to measure TGF-β gene expression in wound tissuesamples of day 7 post wounding.

FIG. 9 show data demonstrating mature collagen deposition in ischemicwounds treated with MCG. A: Representative images of formalin-fixedparaffin-embedded (FFPE) wound biopsy sections (5 μm) stained usingMasson's trichrome; B: Representative images from FFPE wound tissuebiopsy sections stained using picrosirius red staining (PRS); and C:Collagen type I gene expression in day 7 ischemic wound tissues wasquantified using real-time PCR.

FIG. 10 shows data from Example 2 including A: a representative scatterplot of cells harvested from sponges at day-3 post implantationsubjected to flow cytometry analysis and gated for quantification; andB: a graph of FITC-conjugated F4/80 positive cells quantified from thegated cell populations. Data are mean±SD (n=6); *p<0.05 compared tountreated cells.

FIG. 11 shows representative four-quadrant dot plots (column 1 & 2),histograms of FITC+ and PE+ cells (column 3), and quantitative resultsexpressed as MFI of double positive cells (column 4) in the earlyinflammatory phase of wound healing from Example 2. Data are mean±SD(n=6); *p<0.05.

FIG. 12 shows representative four-quadrant dot plots (column 1 & 2),histograms of FITC+ and PE+ cells (column 3), and quantitative resultsexpressed as MFI of double positive cells (column 4) in the lateinflammatory phase of wound healing from Example 2. Data are mean±SD(n=6); *p<0.05.

FIG. 13 shows graphs indicating up-regulation of IL-4(A&B) andIL-10(C&D) gene expression & protein production in mouse inflammatorycells collected from MCG-treated sponges at different time points.

FIG. 14 shows graphs indicating up-regulation of IL-10(A&B) andVEGF(C&D) gene expression and protein production in THP-1 differentiatedhuman macrophages treated with MCG for 72 h.

FIG. 15 shows A: Representative images showing harvested MCG-treatedmacrophages (green, CD68) cultured with apoptotic thymocytes (red, CMTMRcell tracker); B: efferocytosis scoring of thymocytes engulfed bymacrophages, calculated as total number of apoptotic cells engulfed bymacrophages in a field of view divided by total number of macrophagepresented in the view; and C: Mir-21 expression in mouse inflammatorycells collected from MCG-treated sponges at day 3 post implantation,presented as % change compared to untreated cells.

FIG. 16 shows graphs of A: IL-10 production in miR-000-zip or miR-21-zipcells after treatment with MCG; and B: IL-10 production by MCG indifferentiated THP-1 cells pre-treated with anti-TLR-4 antibody,Herbimycin, or vehicle.

FIG. 17 illustrates a process flow for the MCG signaling process throughthe miR-21 pathway.

DETAILED DESCRIPTION

The present invention is concerned with compositions and methods fortreating ischemic wounds, as well as inflammatory conditions. Thecompositions comprise a modified collagen gel. Exemplary modifiedcollagen gels include Stimulen™ (Southwest Technologies, Inc., NorthKansas City, Mo.). In general, the modified collagen gel comprises adispersion of modified collagens in a glycerine or other biocompatiblematrix. In one or more embodiments, the collagen gel comprises modifiedcollagen of long and short polypeptides dispersed in an aqueous matrixcomprising (consisting essentially or even consisting of) water andglycerine. The collagen gel comprises at least about 2% by weightmodified collagen, preferably from about 2% to about 75% by weightmodified collagen, more preferably from about 25 to about 75%, and insome cases, preferably about 52% by weight modified collagen, based uponthe total weight of the composition taken as 100% by weight. Thecollagen gel comprises at least about 15% by weight glycerine,preferably from about 15% to about 65% by weight glycerine, morepreferably from about 25% to about 65%, and in some cases, preferablyabout 45% by weight glycerine, based upon the total weight of thecomposition taken as 100% by weight. In some embodiments, the modifiedcollagen can first be provided in a dry (powdered form), which can thenbe dispersed into a matrix carrier, such as glycerin and/or water beforeuse. In one or more embodiments, the collagen is a hydrolyzed bovinecollagen. In one or more embodiments, the collagens comprise primarilyType I and Type III collagens (and mainly Type I). Specific proteomiccomponents of the preferred modified collagen gel are provided in theTable below.

TABLE 1 Proteomic Analysis of MCG Components * Number of Sl. UnigeneMass significant No Description Accession ID (Da) sequences Score 1Hemoglobin subunit beta HBB_BOVIN Bt.23726 16001 7 685 2 Carbonicanhydrase 2 CAH2_BOVIN Bt.49731 29096 10 650 3 Collagen alpha-1 (1)chain CO1A1_BOVIN Bt.23316 139880 3 321 4 Hemoglobin subunit alphaHBA_BOVIN Bt.10591 15175 5 319 5 Peroxire doxin-2 PRDX2_BOVIN Bt.268922217 5 308 6 Alpha-1-antiproteinase A1AT_BOVIN Bt.982 46417 2 220 7Serpin A3-7 SPA37_BOVIN Bt.55387 47140 3 161 Bt.92049 8 Collagenalpha-1(III) chain CO3A1_BOVIN Bt.64714 93708 2 147 9 Collagenalpha-2(I) chain CO1A2_BOVIN Bt.53485 129499 2 103 10 Serpin A3-3SPA33_BOVIN Bt.55387 46411 2 85 Bt.92049 11 Actin, aortic smooth muscleACTA_BOVIN Bt.37349 42381 2 79Top ten most abundant proteins as detected using proteomics analysis hasbeen presented Two unique peptides from one protein having a -b or -yion sequence tag of five residues or better were accepted. *FromElgharably et al., A modified collagen gel enhances healing outcome in apreclinical swine model of excisional wounds, 21 Wound Repair andRegeneration 473-481 (May-June 2013), incorporated by reference herein.

Compositions of such modified collagen gels have surprisingly been foundto be useful in treating, repairing, promoting the healing of, and/orincreasing the rate of healing of ischemic wounds and other inflammatoryconditions (including dermal and non-dermal conditions). The term“wound” is used herein to refer to injury or disruption to living tissuecaused by a lesion, cut, blow, or other impact in which the skin is cutor broken, as well as to incisions into the skin. Thus, the termencompasses incisions, cuts, lacerations, burns, avulsions, necrosis,and the like of the skin (epidermis), and can be caused accidentally orpurposefully (e.g., such as through surgery). “Ischemic” wounds refer towounds to which the flow of blood has been obstructed, restricted, orotherwise impaired, such that the wound site is deprived of oxygen,nutrients, etc. Damaged tissue deprived of adequate blood flow has adecreased ability to heal, and as such predisposes individuals to thedevelopment of chronic wounds. Unlike acute wounds, chronic ischemicconditions do not heal according to the normal wound healing processinvolving, inter alia, resolution of inflammation, repair of theconnective tissue matrix, and angiogenesis. Accordingly, treatmentprotocols for acute wounds are often ineffective (and in some cases notrecommended) for ischemic or other non-healing chronic wounds. It hassurprisingly been found that the modified gel compositions promote woundhealing even in ischemic wounds.

The compositions are useful in “treating” ischemic wound conditions andother inflammatory conditions, meaning that the composition can beapplied to the site of the wound of a patient or administered (topicallyor injected) to a patient suffering from an inflammatory condition forthe purpose of diminishing or eliminating signs, symptoms, or severityof the wound or condition.

In general, the methods comprise applying or administering atherapeutically effective amount of the composition to the site of thewound or to the patient having the inflammatory condition for atherapeutically effective period of time. In one or more embodiments,the composition is applied as a dressing to the site. The compositionand/or dressing may be changed periodically, wherein a fresh amount ofcomposition is applied to the site. Additionalphysiologically-acceptably non-occlusive dressings, tape, gauze,bandages, combinations thereof, and the like may be used in conjunctionwith the composition, according to standard wound care protocols. Asused herein, the term “therapeutically effective” refers to the amountand/or time period that will elicit the biological or medical responseof a tissue, system, animal or human that is being sought by aresearcher or clinician, and in particular elicit some desiredtherapeutic effect. For example, in one or more embodiments,therapeutically effective amounts and time periods are those that reduceinflammation and initiate or promote healing of the wound site or siteof dermal inflammation. One of skill in the art recognizes that anamount or time period may be considered therapeutically effective evenif the wound or condition is not totally eradicated but improvedpartially. In one or more embodiments, a therapeutically effectiveamount refers to application of the modified collagen gel composition tothe wound site to provide a light coating (e.g., 1/16 inch) up to about⅛ inch of the composition or more, over the wound. The composition canbe changed or re-applied daily, or multiple times per day. Likewise, thecomposition can be applied every other day, every three days, etc. It isnoted that although conventional treatment protocols may call forpacking deep wounds, it is not necessary to fill a deep wound cavitywith the modified collagen gel, and the wound surfaces can simply becoated with the modified collagen gel, followed by packing the woundwith a passive wound dressing to keep pressure on the wound and preventthe modified collagen gel composition from being inadvertently wipedaway from the wound site. Those skilled in the art will appreciate thattreatment protocols can be varied depending upon the wound, healingstatus, and preference of the medical practitioner.

In one or more embodiments, the methods are effective in resolvinginflammation in an ischemic wound or site of dermal inflammation. Themethods involve applying the composition to the wound site or site ofinflammation, wherein the composition actively up-regulates macrophagefunction at the site to initiate the healing process(neo-vascularization), followed by timely up-regulation ofanti-inflammatory factors (e.g., anti-inflammatory cytokines) to resolveinflammation and transition the in vivo healing process to angiogenesis.The methods also involve varying (e.g., increasing) the ratio ofcollagen type I to collagen type III at the wound site or site of dermalinflammation, comprising applying the composition to the wound site orsite of inflammation. Accordingly, the methods are also effective forincreasing the tensile strength of the repaired tissue site therebyreducing or minimizing the incidence of wound recurrence.

The compositions and methods are also useful for enhancing tissueperfusion at the wound site or site of inflammation by applying thecomposition to the wound site or site of inflammation, wherein bloodflow is increased at the site.

The compositions and methods are also useful in resolving otherinflammatory conditions.

Advantageously, the compositions, methods, and treatment protocols canconsist of use of only the collagen gel in the treatment of the ischemicwound or inflammatory condition. In other words, no other adjunctivetherapy is required to initiate or promote healing. As such, in someembodiments, the only therapeutic or “active” agent used in treating thewound or condition is preferably the modified collagen gel composition.No other antibacterial compositions, ointments, hydrogels, therapeuticdressings, and the like are needed, and can preferably be avoided undertypical circumstances. Notwithstanding the foregoing, it will beunderstood that the methods and treatment protocols would stillencompass the use of passive wound care items, such as non-occlusivebandages and gauze, etc. that can be used to cover the treated wound orinflammatory condition once the modified collagen gel has been appliedor administered.

Additional advantages of the various embodiments of the invention willbe apparent to those skilled in the art upon review of the disclosureherein and the working examples below. It will be appreciated that thevarious embodiments described herein are not necessarily mutuallyexclusive unless otherwise indicated herein. For example, a featuredescribed or depicted in one embodiment may also be included in otherembodiments, but is not necessarily included. Thus, the presentinvention encompasses a variety of combinations and/or integrations ofthe specific embodiments described herein.

As used herein, the phrase “and/or,” when used in a list of two or moreitems, means that any one of the listed items can be employed by itselfor any combination of two or more of the listed items can be employed.For example, if a composition is described as containing or excludingcomponents A, B, and/or C, the composition can contain or exclude Aalone; B alone; C alone; A and B in combination; A and C in combination;B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certainparameters relating to various embodiments of the invention. It shouldbe understood that when numerical ranges are provided, such ranges areto be construed as providing literal support for claim limitations thatonly recite the lower value of the range as well as claim limitationsthat only recite the upper value of the range. For example, a disclosednumerical range of about 10 to about 100 provides literal support for aclaim reciting “greater than about 10” (with no upper bounds) and aclaim reciting “less than about 100” (with no lower bounds).

EXAMPLES

The following examples set forth methods in accordance with theinvention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

Example 1 A Modified Collagen Gel Dressing Promotes Angiogenesis in aPreclinical Swine Model of Chronic Ischemic Wounds

Introduction

The porcine model is widely accepted as an excellent preclinical modelfor human skin wounds. In this study, we utilized a well-characterizedporcine model of chronic ischemic wounds. (Roy S. et al.Characterization of a preclinical model of chronic ischemic wound.Physiol Genomics 2009; 37: 211-24). Collagen is the major constituent ofthe dermal extracellular matrix (ECM). In addition to providingstructural support, collagen dressings support granulation tissueformation by enhancing cellular chemo-attraction, differentiation, andactivation. Excessive activity of proteolytic enzymes in chronic woundsthreatens wound closure by degrading ECM proteins and other bioactiveproteins such as growth factors. Clinical application of collagen-basedproducts helps manage excessive proteolysis at the wound site favoringhealing. The current work builds on our recent report characterizing amodified collagen gel (MCG) dressing for wound care. We have earlierreported that MCG improves wound closure in acute excisional wounds.Tissue ischemia is a critical component of chronic wound pathology. Inthe current report, we tested the effect MCG on wound angiogenesis usingan established preclinical porcine model of experimental chronicischemic wound.

Materials and Methods

Porcine Ischemic Flap Model

All experiments were approved by The Ohio State University'sInstitutional Laboratory Animal Care and Use Committee. A total of sixdomestic Yorkshire pigs (70-80 lbs) (Hartley Farms, Circleville, Ohio)were used in this study. Pigs (70-80 lbs) were sedated by Telazol(Zoetis, Florham Park, N.J.) and anesthetized by mask with isoflurane(3-4%). The dorsal region was shaved, and the skin was surgicallyprepared with alternating chlorhexidine 2% and alcohol 70% (ButlerSchein, Columbus, Ohio) scrubs. Four full-thickness bipedicle skin flapsmeasuring 15×5 cm were created on each animal as described by Roy et al.Sterilized silicone sheets (15×5 cm) (Technical Products Inc., Decatur,Ga.) were placed underneath the flaps, and then the flap and siliconesheet edges were sutured to the adjacent skin. Laser Doppler scanning ofthe flaps was used to verify blood flow status and degree of ischemia.

Wounding and Treatments

A full-thickness excisional wound was created in the center of each flapusing an 8-mm disposable biopsy punch. The depth of the wound wasmeasured by the length of stainless steel section of the punch biopsy (8mm). The wounds were created by cutting through the skin until theentire length of the stainless steel section was below the skin and theplastic shoulders (edges) of the biopsy punch were at the surface of theskin. That length was adequate to reach the subcutaneous fat in allwounds. Flap edges were sutured to adjacent skin and the underlyingsilicone sheet to prevent revascularization from the sides or underneathExcisional wounds on one side were treated with an MCG followed bydressing with Tegaderm™ (3M, St. Paul, Minn.). The wounds in thecontralateral flaps were covered with Tegaderm™ alone as standard ofdressing care (control). Treatment sides were alternated between animalsto avoid any side-specific effect. All four flaps were covered withV.A.C. Drape (Owens & Minor, Mechanicsville, Va.). Dressing was changedevery 5-7 days, and any accumulating wound fluid was drained as needed.On designated time points (days 7 and 21 post wounding), the entirewound tissue was harvested using a 10-mm biopsy punch, and each samplewas split into three pieces for subsequent analyses:immunohistochemistry (OCT frozen) studies, RNA studies, and formalinfixed histology studies. Animals were maintained on 12-hour light-darkcycles and were euthanized after the completion of experiments.

MCG was provided as Stimulen™ gel by Southwest Technologies Inc. (NorthKansas City, Mo.). According to the manufacturer, the unique formulationof the MCG represents a mixture of 52% collagen of long and shortpolypeptides along with glycerine, water, and fragrance. The MCG is ahighly concentrated modified collagen (mainly type I) in a gel form.

Laser Doppler Scanning of Blood Flow

The MoorLDI-Mark 2 laser Doppler blood flow scanner (Moors Instruments,Axminster, United Kingdom) (resolution: 256×256 pixels in the region ofinterest; each pixel being an actual measurement) was used to studytissue perfusion. Laser Doppler scanning was performed after thesurgical procedure and at day 21 post wounding.

Histology

Formalin-fixed paraffin-embedded or optimum cutting temperature-embeddedfrozen wound-edge specimens were sectioned (5 μm). The paraffin sectionswere deparaffinized and stained with hematoxylin & eosin (H&E) (FIG. 1),Masson's trichrome, or picrosirius red staining (PRS) using standardprocedures. FIG. 1 has mosaic images showing MCG treated or TD,Tegaderm™ treated ischemic wounds on day 7 after wounding—HE:hyperproliferative epithelium; FP: fibrin plug; and GRN: granulationtissue.

Immunohistochemical staining of paraffin or frozen sections wasperformed using the following primary antibodies: anti-macrophage, L1calprotectin (1:400; MAC387; Thermo Fisher Scientific Inc., Waltham,Mass.), anti-von Willebrand's factor (vWF) (Dako North America Inc.,Carpinteria, Calif.), anti-Ki67 (1:400, Thermo Fisher Scientific Inc.),anti-vimentin (Sigma-Aldrich, St Louis, Mo.), and anti-CCR2 (1:250;Abcam, Cambridge, Mass.) after heat-induced epitope retrieval whennecessary. Secondary antibody detection and counterstaining wereperformed.

Imaging

Mosaic images of whole wound sections were collected under 20×magnification guided by MosaiX software (Zeiss, Thornwood, N.Y.) and amotorized stage. Each mosaic image was generated by combining 40-50images. Each mosaic image was generated by combining a minimum of 100images. Between 7 and 9 high-powered representative areas from mosaicimages were quantified for each data time point. Image analysis wasperformed by employing auto-measure software (Zeiss) for quantitation ofthe percentage of immuno-histochemical positive areas (expressed as %area). Confocal Scanning Laser Microscope: visualization of vascularstructures within the wound tissues was achieved by using an OlympusFluoview FV1000 spectral confocal microscope (Olympus, Pittsburgh, Pa.)under 1,000× magnification while applying an argon laser. Z-stack imageswere created by merging serial scans of thick tissue section (20 μm).Cell culture Human THP-1 monocytes (American Type Culture Collection,Manassas, Va.) were cultured in RPMI 1640 medium with L-glutaminesupplemented with 10% FBS and 1% antibiotic antimycotic (AA) (Gibco,Auckland, New Zealand) and incubated at 37° C. in 5% CO2. Todifferentiate THP-1 monocytes into macrophages, cells were cultured inRPMI 1640 medium with L-Glutamine supplemented with 10% heat inactivatedFBS, 1% AA, and 20 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma,St. Louis, Mo.). Supplementation THP-1 cells were incubated with orwithout MCG (50 mg/mL) for 24 hours. RNA was extracted from cell pelletsusing mirVana RNA isolation kit (Ambion, Austin, Tex.) according to themanufacturer's instructions.

RNA Isolation from Wound Tissues

Immediately after collection, wound tissue biopsies were rinsed insaline, patted dry, and snap frozen in liquid nitrogen. Grinding of thetissues was performed using a 6770 Freezer/Mill cryogenic grinder (SPEXSamplePrep, Metuchen, N.J.). Total RNA from tissue or cultured cellswere extracted using mirVana RNA isolation kit. Reverse transcriptionand quantitative real-time polymerase chain reaction (PCR) Tissue mRNAwas quantified by real-time or quantitative (Q) PCR assay using thedouble-stranded DNA binding dye SYBR green-I. The primer set used forthe individual genes are described in Elgharably et al., Wound Rep Reg(2014) 22 720-729, incorporated by reference herein. 18s rRNA was usedas a reference housekeeping gene.

Statistics

Data are reported as mean±standard error of the mean of six separateanimals as indicated. As the data were not normally distributed,nonparametric statistics was used. Wilcoxon signed-rank test was used tocompare control vs. MCG as the wounds were paired within the individualpigs. The significance level for this study was set at 0.05. Allanalyses were run using Stata 13.1 (StataCorp, College Station, Tex.).

Results

The preclinical model of chronic ischemic wounds was employed for thisstudy. Full-thickness bipedicle flaps were surgically created on theback of pigs in such a way that interrupted blood supply from underneathand the sides of the flap. After the procedure, ischemia of flap tissuewas confirmed by laser Doppler imaging of blood flow. The full-thicknessexcisional wounds established in the middle of the flap rested on themost poorly perfused part of the flap. By harvesting whole wound tissuesat day 7 and 21 post wounding, we were able to study the effect of MCGon both early and late events of wound healing processes. Successfulmounting of inflammatory response and timely resolution are necessaryfor proper healing of a wound. As part of investigating the inflammatoryresponse, wound macrophages were identified in wound-edge tissuesections using an antibody against macrophage L1 protein/calprotectin(MAC387), a marker for swine tissue macrophages. Anti-MAC387 alsorecognizes other cells such as keratinocytes and polymorphonuclearneutrophils (PMNs). However, in this case, keratinocytes were ruled outusing anti-keratin-14 (keratinocyte specific, data not shown), the cells(MAC387) did not co-localized with K14 positive cells, thus excludingkeratinocytes. For PMN, the nuclear morphology is very distinct ascompared with macrophages. 4′,6-diamidino-2-phenylindole (DAPI; nuclearstain, blue) staining confirmed that most of the MAC387 positive cellswere macrophages.

Macrophage infiltration to the wound-edge tissue was significantlyincreased in ischemic wounds treated with MCG at day 7 post woundingcompared with untreated control wounds (FIGS. 2A and B). FIG. 2A showsrepresentative mosaic images of wound-edge tissues that wereimmunostained using Anti-MAC387 (macrophages, green). The sections werecounterstained with DAPI (blue). Scale bar, 200 Insets are zoomedregions in the image; Scale bar, 50 FIG. 2B shows a bar graph ofquantitation of macrophage infiltration at day 7 post wounding inischemic wounds treated or untreated with MCG. Data are presented asmean±SEM (n=6); *p<0.05 compared with untreated wounds.

This finding directed us to further investigate the effect of MCG onmacrophage function in vitro. THP-1-derived macrophages treated with MCGdisplayed up-regulation of Mrc-1 gene expression, which is a marker for(M2) reparative macrophage subtype (FIG. 2C). The data in FIG. 2Cdemonstrate up-regulation of macrophage mannose receptor 1 (MRC-1) geneexpression in THP-1 differentiated human macrophages treated with MCGfor 24 hour. MRC-1 gene expression was measured using quantitativereal-time PCR.

Furthermore, we observed that MCG potently induced the anti-inflammatorycytokine IL-10 and the fibroblast growth factor basic (β-FGF) geneexpression (FIG. 3). To validate the in vitro finding of increased M2macrophage marker by MCG, we studied the phenotype of wound macrophagein MCG-treated wounds. The expression of M2 macrophage markers Arg-1 andCCR2 was determined in wound tissues (FIGS. 4A and B). Real-time PCR wasused to measure Arg-1 and CCR2 gene expressions in wound tissue samplesat day 7 post wounding. Gene expression data are presented as % changecompared with untreated wound tissues. Data are mean±SEM; *p<0.05. Amassive induction in Arg-1 and CCR2 was noted in wound tissue followingMCG treatment as compared with standard of care (TD, Tegaderm™). Recentstudies indicate CCL2-CCR2 axis plays a major role in shaping macrophagepolarization. In M2c macrophages, IL-10 increases expression of CCR2 andCCR5. An immunohistochemistry co-localization study was performed withanti-L1 (Macrophage, green) and anti-CCR2 (M2c macrophage, red) (FIG.4C). The sections were counterstained with DAPI (blue). Scale bar, 10μm. Insets are zoomed regions in the image, Scale bar, 1 Increased CCR2positive macrophages in wound tissue 7 days post wounding was observedsuggesting that either promotion of M2 macrophages recruitment orincreased conversion of wound site macrophages to M2 phenotype. In vitrodata showing a direct effect of MCG on macrophages by increasingexpression of M2 marker supports the latter proposition, i.e., increasedconversion to M2 phenotype by MCG. One of the potent angiogenic factorsthat promote wound revascularization is vascular endothelial growthfactor (VEGF). We observed significant increase of VEGF gene expressionat day 7 post wounding in wound-edge tissue treated with MCG comparedwith corresponding control (FIG. 5A). In concordance with thatobservation, higher abundance of endothelial cell marker vWF wasdetected in wound-edge tissues of MCG-treated wounds (FIG. 5B). TotalRNA was isolated from wound-edge tissue material stored in liquidnitrogen. Real-time PCR was used to measure VEGF and vWF geneexpressions in samples at day 7 post wounding. Gene expression data arepresented as % change compared with untreated wound tissues. Data aremean±SEM (n=6); *p<0.05. Histological quantitation of endothelial cellsabundance in wound-edge tissues showed a markedly elevated count inwounds treated with MCG reflecting increased endothelial cellproliferation (FIG. 5C). Scale bar, 500 Lower panel are zoomed regionsin the images on top; Scale bar, 50 Negative control image showsspecificity of the anti-vWF staining. The section was counterstainedwith DAPI (blue, nuclear) to show the cells present in the section. Bargraph shows quantitation of the endothelial cells in MCG-treated oruntreated ischemic wounds at day 21 post wounding. Data are presented asmean±SEM (n=6); *p<0.05 compared with untreated wounds. White dashedlines indicate the edges of the wound. TD, Tegaderm™; MCG.

MCG-treated ischemic wounds also had markedly higher abundance ofendothelial cells 21 days post wounding indicating that the favorableeffect of MCG on endothelial cell proliferation was persistent. Dualimmunofluorescence staining technique was employed to co-localizecell-proliferation marker Ki67 within endothelial cells. Ki67 is anuclear protein that is associated with cellular proliferation.Quantitative analysis of Ki67 in vWF+ endothelial cells using anautomated software-based methodology revealed that MCG treatmentmarkedly enhanced endothelial cell proliferation at the ischemicwound-edge tissue (FIG. 6). A marked increase of proliferatingendothelial cells in MCG-treated ischemic wounds compared with controlwounds as evident by co-localization of Ki67 within vascular structures(yellow areas in merged images) was noticed. The bottom panels arezoomed regions within the dashed white boxes in the corresponding middlepanels. Top and middle panels scale bars, 50 μm. Bottom panel scale bar,20 μm.

Next, we sought to address the quality and functionality of vascularstructures at the wound edge. Thick wound-edge tissue sections (20 μm)were immuno-stained for vWF and examined using confocal laser scanningmicroscope (CLSM). Qualitative analyses of wound-edge vascularstructures were performed using CLSM by creating Z-stack merged images.Interestingly, on day 21 post wounding, MCG-treated ischemic woundsdisplayed more mature and thick vascular formations, whereas controlwound-edge tissue featured a scanty distribution of thin vascularstructures (FIG. 7A). Note increased mature vascular structures inMCG-treated ischemic wounds compared with untreated wounds in the x/yplane, whereas the x/z and y/z planes display the thickness of thevascular structures in the tissue section.

To determine blood flow, we applied laser Doppler imaging technology.Laser Doppler scanning is a reliable and applicable method that providesinformation about tissues' perfusion without physical contact. At day 21post wounding, scanning of flap tissues by laser Doppler showedsignificant increase of blood flow to wound tissue treated with MCGcompared with corresponding control wounds (FIG. 7B). Marked increase inblood flow (red) was noted in ischemic wounds treated with MCG. Bargraph represents the quantitative data from laser Doppler analysis. Datapresented as mean±SEM (n=6); *p<0.05 compared with untreated wounds.

Collectively, MCG-treated ischemic wounds had higher abundance ofproliferating endothelial cells that formed mature and functionalvascular structures with an increase of blood flow to the wound site.Fibroblast proliferation is a key driver of the proliferative phase ofwound healing. Fibroblasts synthesize collagen and other ECM componentsthat form the scaffold necessary for the migration and proliferation ofother cell types involved in wound repair. Vimentin has been usedroutinely as a marker for dermal fibroblasts. Vimentin is one of theintermediate filaments expressed in cells of mesenchymal origin. Othercells of mesenchymal origin such as pericytes may express vimentin.Immunofluorescence staining data revealed that vimentin expression inMCG-treated ischemic wounds was significantly higher on day 21 postwounding compared with untreated wounds (FIGS. 8A and B). White dashedlines indicate the edges of the wound. Middle and right panels are zoomsof the boxed areas within the images in the left panels. Scale bar, 100μm (middle); Scale bar, 10 μm. Data are presented as mean±SEM (n=6);*p<0.05 compared with untreated wounds. Furthermore, on day 7 postwounding, wound-edge tissue samples from MCG-treated group showedup-regulation of TGF-β gene expression compared with correspondingcontrol wounds (FIG. 8C). Gene expression data are presented as % changecompared with untreated control wound tissues. Data are mean±SEM (n=4);*p<0.05. TD, Tegaderm™; MCG.

TGF-β is one of the regulatory growth factors that contribute tofibroblasts' recruitment and stimulation of collagen expression inhealing wounds. These two findings, along with cyto-morphologicalproperties of vimentin positive structures, point to the conclusion thatMCG-treated ischemic wounds were heavily populated by fibroblasts on day21 post wounding. Appropriate collagen deposition helps build tensilestrength of the nascent tissue. Histological characterization ofcollagen in pair-matched wounds was performed through Masson's trichromeand PRS. Masson's trichrome staining of wound-edge tissues showed higherdeposition of mature collagen fibers in MCG-treated ischemic wounds(FIG. 9A). This staining results in blue-black nuclei, blue collagen,and light red or pink cytoplasm. Epidermal cells appear reddish. Scalebar, 200 μm. Black dashed lines indicate the edges of the wound. Rightpanels are the zooms of the boxed areas within the images in the leftpanels. Scale bar, 100 μm. Bar graph shows quantitation of collagenabundance in MCG treated or -untreated ischemic wounds on day 21 postwounding. Data are presented as mean±SEM (n=6); *p<0.05 compared withuntreated wounds. PRS was used to identify both types I and III collagenfibers at the wound site. In tissues stained with PRS, type I collagen(thick fibers) is viewed as yellow orange birefringence, whereas typeIII (thin fibers) is viewed as green birefringence under polarized lightmicroscope. Significant increase of collagen type LIII deposition wasnoted in MCG-treated wounds (FIG. 9B). This stain can be used todistinguish between type I and type III collagen in wound tissues; typeI (thick fibers) appears yellow-orange birefringence, whereas type III(thin fibers) appears green birefringence when viewed under polarizedlight microscope. Showing is a marked increase of collagen LIII ratio(yellow-orange fibers to green fibers) in MCG-treated ischemic woundscompared with untreated control wounds. Scale bar, 100 μm. Thishistological phenotypic change was supported by up-regulation ofcollagen type I gene expression in MCG-treated ischemic wounds in day 7wound tissue samples (FIG. 9C). Gene expression data are presented as %change compared with MCG-untreated control wound tissues. Data aremean±SEM (n=6); *p<0.05.

Discussion

Management of ischemic tissue ulceration represents a substantialclinical challenge. Excessive proteolysis at the wound site results inuncontrolled degradation of the ECM and growth factors that areessential for normal tissue repair. From a clinical point of view,collagen-based products are safe and easily applicable wound dressingsthat can be combined with other modalities. The mechanism of action ofcollagen-based products is poorly understood. Exogenous collagen towounds has been shown to promote hemostasis and chemotaxis. In addition,collagen dressings act as matrices for new cell ingrowth. The currentreport for the first time shows efficacy of MCG, a collagen-baseddressing, in the harsh environment of ischemic wounds by resolvinginflammation and promoting wound angiogenesis. Failure to resolveinflammatory state in a timely manner eventually leads to tissuenecrosis with increased risk of serious complications such as secondaryinfection and amputation. A unique feature of collagen-based products isthat they are effective in managing excessive proteolytic enzymeactivity. Furthermore, exogenously added collagen is claimed toreplenish the wound site ECM, thereby providing a scaffold for cellrecruitment and migration. Interactions between the ECM molecules andcell surface receptors regulate cellular and molecular events of theproliferative phase including epithelialization, angiogenesis, andfibroplasia.

Wound site macrophages represent key drivers of wound repair in theinflammatory phase. Monocytes recruited to injury site differentiate topro-inflammatory macrophages (M1) or anti-inflammatory/pro-angiogenicmacrophages (M2). The M1 macrophages are responsible for clearing ofinfectious agents through secretion of pro-inflammatory cytokines andchemokines that stimulate the immune response. A switch frompro-inflammatory to anti-inflammatory macrophage phenotypes occursfollowing engulfment of apoptotic inflammatory cells also known asefferocytosis. M2 macrophages help resolve inflammation in a timelymanner and induce granulation tissue formation through enhancing ECMsynthesis, angiogenesis, fibroblast proliferation, andepithelialization. Imbalance between pro- and anti-inflammatory signalsin the direction of the former results in persistent wound inflammationand failure to enter the reparative phase of healing. Higher M1:M2macrophage ratio results in wound chronicity. Our recent work shows thatmacrophage dysfunction with persistent pro-inflammatory signaling isresponsible for chronicity of diabetic wounds. MCG enhanced macrophagerecruitment to the ischemic wound site in the early phase is indicativeof a strong inflammatory response. Mrc-1 (mannose receptor c) expressionis recognized as a marker for the M2 macrophage phenotype. IncreasedCCR2 is a marker for IL-10-induced M2c macrophages.

We recognize that increased expression of only one M2c macrophage (CCR2)in MCG-treated wounds does not conclusively show that MCG promotes M2macrophage switching. However, this evidence along with in vitro findingon isolated macrophage (Mrc-1) and increased expression of M2 macrophagemarkers (Arg-1 and CCR2) strongly suggests a potential role of MCG inmacrophage polarization in wounds. IL-10 is a major anti-inflammatoryagent that helps execute scar-minimized regenerative healing of fetalwounds. 0-FGF is a key growth factor that promotes granulation tissueformation and wound closure through stimulation of wound angiogenesis,fibroblasts proliferation, and migration. Ischemic wounds show delayedmacrophage recruitment to the wound site. Taken together, the currentstudy shows that even under conditions of ischemia, MCG promotedmacrophage recruitment to wounds. Furthermore, presence of MCG helpedswitching of M1 macrophages to M2 phenotype suggesting MCG not onlyincreases macrophage recruitment but also helps in resolution ofprolonged inflammation, a characteristics of ischemic wounds.

Ischemic wounds lack blood-borne products such as oxygen, nutrients, andcirculating cells that are necessary for the tissue repair process.Enhancing tissue perfusion therefore represents a useful strategy torescue ischemic wounds. A robust inflammatory response is known to drivewound neovascularization. In this context, wound site macrophages play amajor role. Macrophages secrete pro-angiogenic factors such as VEGF.Signaling between endothelial cell surface receptors and ECM molecules,such as collagen, stimulates migration and proliferation of endothelialcells. Also, it has been shown that the three-dimensional structure ofthe collagen matrix helps endothelial cells to organize into maturevascular structures. An abundance of proliferating endothelial cellsassociated with mature capillary-like structures in MCG-treatedwound-edge tissue is indicative of a potent effect of the dressing onwound vascularization. This contention is supported by improved woundsite blood flow data. Collagen deposition at the site of healingempowers the nascent tissue with tensile strength that helps preventwound reopening. Collagen deposition is known to be inadequate inischemic wounds that accounts for wound dehiscence and failure to close.At the wound site, collagen synthesis is primarily contributed byfibroblasts. Of the several factors that determine the recruitment andproliferation of fibroblasts to the wound site, ECM and growth factorsrepresent major components. Transforming growth factor-β (TGF-β)promotes fibroblast migration, proliferation, and collagen production.Increased expression of TGF-β together with increased fibroblastabundance in MCG-treated ischemic wounds suggested increased collagensynthesis in these wounds. Indeed, abundant mature collagen fibers wereidentified in MCG treated. Importantly, collagen type I dominated overcollagen type III. Such increased collagen type LIII ratio is crucialfor appropriate wound tensile strength to support the growth ofvascularized granulation tissue and to prevent dehiscence. In summary,this study provides novel insight into the mechanism of action of acollagen-based dressing as it relates to outcomes of experimentalischemic wounds. Earlier we showed efficacy of collagen-based MCGdressing improved inflammatory cell infiltration and angiogenesis inacute excisional wound. Using a porcine ischemic wound model, we havereported that such wounds exhibit prolonged inflammatory phase and poorangiogenesis. The current study shows that even under conditions ofischemia, i.e., oxygen and nutrient deprivation, MCG is effective inbolstering a reparative inflammatory response followed by improved woundvascularization and favorable organization of the ECM. Taken together,the observations of the current study warrant testing the efficacy ofMCG in a clinical setting.

Example 2

A Collagen Gel Based Wound Dressing Resolves Inflammation Through amiR-21 Dependent M2 Macrophage Polarization

Reference is made to FIGS. 10-17. The data indicates that MCG recruitshigher levels of initial macrophage response to the site ofinflammation. MCG also facilitates resolution of inflammation sooner inthe healing process by signaling the switch of pro-inflammatorymacrophages to reparative macrophages faster than untreated sites. Thedata supports potential use of MCG in a wide variety of inflammatoryconditions, and that it may have beneficial impact on a wide variety ofpathways for inflammatory disorders.

In FIG. 10, cells were harvested from sponges pre-treated with MCG onDay 3 post implantation subjected to flow cytometry analysis and gatedfor quantification. The FITC-conjugated F4/80 positive cells werequantified from the gated cell populations. Data are mean±SD (n=6);*p<0.05 compared to untreated cells. As shown in FIG. 10, based uponflow cytometry analysis, the data indicates that MCG recruits higherlevels of macrophages to the wound (initial and pro-inflammatory M1macrophage stages) earlier in the inflammatory process.

In FIG. 11, cells were harvested from sponges pre-treated with MCG onDay 3 and subjected to flow cytometry analysis and gated forquantification. Data are mean±SD (n=6); *p<0.05 compared to untreatedcells. The data indicates that although MCG recruits higher levels ofwound macrophages to the wound (initial and pro-inflammatory M1macrophage stages) early in the inflammatory process, as shown in FIG.10, the level of pro-inflammatory macrophages subsides quicker than theuntreated control, indicative of inflammation levels resolving earlierin the process. As such, there is a lower abundance of Wound MacrophageM1 Phenotype in response to MCG in the Early Inflammatory Phase.

In FIG. 12, cells harvested from pretreated sponges 7 days postimplantation and stained with fluorescent-tagged FITC anti-F4/80 and PEM2 markers, then subjected to flow cytometry analysis. Data are mean±SD(n=6); *p<0.05. The data indicates that during the later stages of theinflammatory process, MCG facilitates a quicker switch from thepro-inflammatory M1 macrophage phenotype to the reparative M2 macrophagephenotype. Higher levels of reparative macrophages are seen in thepresence of MCG, as compared to the controls, in the late inflammatoryphase. Thus, not only does MCG initially recruit a higher macrophageresponse to the inflammatory process, but facilitates and earlier switchto the reparative phenotype, resulting in an earlier start to theresolution of the inflammatory response.

In FIG. 13, the data relates to measurement of anti-inflammatorycytokines (expression and protein levels) in the presence and absence ofMCG. The data shows upregulation of IL-4 and IL-10 gene expression &protein production in mouse inflammatory cells collected fromMCG-treated sponges at different time points. Both genes expression weremeasured using quantitative real-time PCR. Protein production wasmeasured by ELISA. Data are presented as % change compared to untreatedcells. Data are mean±SD (n=6); *p<0.05. The data indicates that thelevel of anti-inflammatory cytokines IL-4 and IL-10 are higher in thepresence of MCG as compared to the controls. In particular, the level ofIL-10 expression is ten times higher than the controls at day 3 (earlyin the inflammatory process), while the level of IL-10 protein is aboutsix times higher at day 3.

FIG. 14 shows graphs demonstrating increased IL-10 & VEGF production byTHP-1 derived macrophages after treatment with MCG in vitro, based uponincreases in gene expression and protein production in THP-1differentiated human macrophages treated with MCG for 72 h. Both genesexpression were measured using quantitative real-time PCR. Proteinproduction was measured by ELISA. Data are presented as % changecompared to untreated cells. Data are mean±SD (n=4); *p<0.05.

In vitro work carried out using human THP-1 derived macrophages isillustrated in FIG. 15. The macrophages were incubated with MCG alone.The data demonstrates that MCG bolsters phagocytosis and induces mir-21expression in mouse macrophages. The MCG-treated macrophages (green,CD68) were cultured with apoptotic thymocytes (red, CMTMR cell tracker).Cells were counterstained with DAPI (nuclear, blue). Efferocytosisscoring of thymocytes engulfed by macrophages was carried out andcalculated as total number of apoptotic cells engulfed by macrophages ina field of view divided by total number of macrophage presented in theview. The Mir-21 expression in mouse inflammatory cells collected fromMCG-treated sponges at day 3 post implantation was determined, presentedas % change compared to untreated cells. Data are mean±SD (n=4);*p<0.05. The data indicates that MCG is directly responsible forsignaling the macrophage activity and increasing IL-10 expression andprotein levels. MCG is also responsible for inducing VEGF expression andincreased protein levels as compared to the controls.

In FIG. 16, miR-000-zip or miR-21-zip cells were treated with MCG. Dataare mean±SD (n=4); *p<0.05 compared with non-treated miR-000-zip(control) cells; †p<0.05 compared with treated miR-000-zip cells.Differentiated THP-1 cells pre-treated with anti-TLR-4 antibody,Herbimycin, or vehicle were subsequently treated with MCG. Data aremean±SD (n=4); *p<0.05 compared to non-treated cells; †p<0.05 comparedto cells treated with MCG only. The data illustrates that MCG increasesphagocytosis of dead cells (bottom panel) as compared to the control(image in top panel). Macrophages in the presence of MCG have much moreefficient efferocytosis than the controls. As seen in C, MCG is alsoassociated with increased miR-21 expression, which provides evidencesupporting potential applicability of MCG in a wide variety ofinflammatory conditions.

The data compares effect of MCG in IL-10 production when miR-21 issilenced (miR-21-zip). The data indicates that the activity of MCG isthrough a miR-21-dependent pathway. When miR-21 was silenced, there wasa marked decrease in IL-10. The slight increase over the control in thesilenced test is attributed to only achieving a partial silencing ofmiR-21 (80-90% silenced). Thus, as shown in FIG. 17, MCG signaling isthrough the miR-21 pathway.

We claim:
 1. A method of treating an ischemic wound in a patient, saidpatient having an ischemic wound site said method comprising: topicallyapplying or injecting, for a therapeutically effective period of time, atherapeutically effective amount of a modified collagen gel compositionto said ischemic wound site to yield a treated ischemic wound site,wherein said modified collagen is a hydrolyzed bovine collagen, whereinsaid modified collagen gel comprises a plurality of proteins comprisingHemoglobin subunit beta, Carbonic anhydrase 2, Collagen alpha-1(I)chain, Hemoglobin subunit alpha, Peroxire doxin-2,Alpha-1-antiproteinase, Serpin A3-7, Collagen alpha-1(III) chain,Collagen alpha-2(I) chain, Serpin A3-3, Actin, and aortic smooth muscle,and promotes healing of said treated ischemic wound site.
 2. The methodof claim 1, wherein said modified collagen gel comprises said modifiedcollagen dispersed in an aqueous matrix comprising water and glycerine.3. The method of claim 2, wherein said collagen gel comprises an amountof each of Type I, Type II, and Type III collagen, wherein the amount ofType I collagen is greater than the amount of Type II or Type IIIcollagen, and wherein the amount of Type III collagen is greater thanthe amount of Type II collagen.
 4. The method of claim 2, wherein saidmodified collagen gel comprises from about 25 to about 75% by weight ofsaid modified collagen dispersed in said aqueous matrix, based upon thetotal weight of the gel composition taken as 100% by weight.
 5. Themethod of claim 1, wherein said modified collagen gel promotes healingof said treated ischemic wound site by up-regulating macrophagefunctions of said patient at said treated ischemic wound site.
 6. Themethod of claim 5, wherein said modified collagen gel further resolvesinflammation at said treated ischemic wound site by up-regulatinganti-inflammatory cytokines of said patient at said treated ischemicwound site.
 7. The method of claim 1, wherein said healing comprisesneo-vascularization of said treated ischemic wound site.
 8. The methodof claim 1, wherein said healing comprises angiogenesis of said treatedischemic wound site.
 9. The method of claim 1, said modified collagengel increases the ratio of the patient's collagen type I to collagentype III at the ischemic wound site.
 10. The method of claim 1, furthercomprising covering said treated ischemic wound site with anon-occlusive bandage, gauze, tape, or a combination thereof.
 11. Themethod of claim 1, wherein said modified collagen gel comprises adispersion of about 52% by weight of said hydrolyzed bovine collagen,dispersed in an aqueous matrix comprising water and glycerine.